In order to understand the function of the human genome, knowing the genome sequence alone is not sufficient. We also need to know the physical 3D path of all DNA molecules (chromosomes) of the genome within the nucleus of a cell. For essential genome activities, e.g. transcription, replication, and its transmission to the next generation in cell division, it is furthermore necessary to understand how the 3D architecture dynamically changes. Insights at the single cell level from conventional light microscopy have so far remained at the superficial level of whole chromosome territories since the critical genome structure elements of topologically associated domains (TADs) and their connecting fibers lie below the diffraction limit of light. Furthermore, we have very little dynami knowledge since sequence-specific labeling in live cells has been difficult, and chromatin dynamics are particularly light sensitive. Two technological breakthroughs, i.e. the advent of super-resolution microscopy (SRM) with a resolution of a few nucleosomes combined with novel computational data analysis algorithms, and the ability to label any DNA sequence of interest fluorescently in living cells by genome editing- based tools, now make it possible to address this fundamental barrier to our progress. Here, we propose to develop 3D and 4D SRM technologies to enable us to determine the 3D structure of stable chromatin domains, resolve how such domains are interconnected and organized in 3D to form a chromosome, and monitor the structurally dynamic DNA sequences in real time during cell division. We will provide (1) realistic computer simulations of all chromatin fibers in the nucleus, reconstruction algorithms which can transform a series of SRM- generated probe positions into the 3D chromatin path; (2) new labeling strategies to label the entire genome with 10 kb resolution with SRM-compatible probes in ~10 discernible colors, and a method to label multiple specific loci in living cells in two colos; and (3) multi-color supercritical angle and inverted lattice light-sheet microscopy with ~20 nm and ~30 nm resolution, respectively, with high-throughput sample and live cell handling. Integration of these computational, experimental, and imaging technologies will result in an integrated workflow for 3 and 4D, robust, and fully automated genome structure analyses. We will then use this workflow to analyze the genome on three structural levels: individual TADs, TAD clusters, and whole chromosomes, and follow structural changes of whole chromosomes during the cell cycle by live cell 3D super-resolution microscopy. The resulting data will be a breakthrough for our scientific knowledge - the first map of the 3D path of the linear genome sequence in the nucleus of a single human cell and the first characterization of how this 3D organization changes throughout the cell cycle. Reliable imaging technology to determine the genome structure of single cells will be invaluable for all fields of genome biology as well as for cell cycle and mitosis research, and offers many exciting possibilities for clinical applications t better understand and diagnose diseases associated with genome instability such as cancer.